The universal donor for specific selenium incorporation into proteins has been identified as monoselenophosphate. Selenophosphate synthetase catalyzes the formation of this reactive selenium donor compound from selenide and ATP. In recent years, the catalytic mechanism of the selenophosphate synthetase enzyme from Escherichia coli has been studied extensively. The enzyme has a very low determined specific activity for its catalyzed reaction (83 nmol/min/mg). We speculated that the reason for this low in vitro specific activity may be that free selenide is not the actual substrate for the enzyme. To address this issue we obtained the NIFS protein from Azobacter vinelandii from Dennis Dean and used it as a selenide delivery protein along with selenocysteine as its substrate in our in vitro selenophosphate synthetase assay. The results of our coupled assays demonstrated that the selenium released from selenocysteine by NIFS can be used as a substrate by selenophosphate synthetase. In fact, the rate of selenophosphate formation by the coupled NIFS selenophosphate synthetase system was higher than that observed in selenophosphate synthetase assays performed with selenide added directly. We postulate that in vivo a more specific selenide delivery protein is involved in the delivery of selenide to enzymes, such as selenophosphate synthetase, which utilize it as a substrate. Additional research has been focused on the enzymatic mechanism of selenophosphate synthetase. Recently, homologs from other organisms have been identified. Sequence comparison of all identified homologs revealed a highly conserved glycine-rich sequence in the N-terminal region of the protein. The E. coli homolog contains a cysteine at position 17 in this region, which has been identified as an essential residue by mutagenesis. Five of the homologs contain a selenocysteine residue at the position corresponding to cysteine 17 in the E. coli enzyme. To understand the role of selenocysteine in these proteins, the selenocysteine-containing selenophosphate synthetase from Haemophilus influenzae was cloned and overexpressed in E. coli. The recombinant protein contained selenocysteine at the expected residue and was kinetically characterized for direct comparison with the cysteine-containing E. coli enzyme. Analysis revealed the H. influenzae enzyme does not have increased catalytic activity. This result suggests selenocysteine may not be involved in a catalytic function of this enzyme. Additional mutagenesis is now being performed to better understand the function of this residue.